氧化应激诱导内皮细胞凋亡microRNA表达改变的研究

目的:研究氧化应激诱导的内皮细胞micro RNA的表达变化。方法:ECM(Endothelial Cell Medium)培养人脐静脉内皮细胞,利用不同浓度双氧水(0μmol/L,200μmol/L,500μmol/L,800μmol/L)刺激24小时后应用流式细胞术检测其凋亡水平。提取细胞总RNA,利用实时定量PCR(Quantitive real-time PCR;q RT-PCR)检测micro RNA表达量变化,并利用生物信息学软件预测可能的靶基因。结果:加入不同浓度双氧水处理24 h后的内皮细胞总凋亡率均显著高于对照组,200μmol/L、500μmol/L和800μmol/L组的凋亡率分别为(13.31%vs 4.75%,35.9%vs 4.75%,89.75%vs 4.75%,P0.01)。200μmol/L的双氧水处理内皮细胞后,micro RNA的表达出现了明显的改变。其中mi R-92a、mi R-126的表达明显下调(P0.05),mi R-181a、mi R-217、mi R-34a和mi R-320的表达明显上调(P0.05)。靶基因预测显示mi R-320、mi R-92a可能调控多个和内皮细胞凋亡相关的基因表达。结论:在氧化应激诱导的内皮细胞凋亡中,mi RNA表达发生改变并可能参与调控内皮细胞功能。     

Pitavastatin Strengthens the Barrier Integrity in Primary Cultures of Rat Brain Endothelial Cells

Statins have a neuroprotective effect in neurological diseases, a pleiotropic effect possibly related to blood–brain barrier (BBB) function. We investigated the effect of pitavastatin on barrier functions of an in vitro BBB model with primary cultures of rat brain capillary endothelial cells (RBEC). Pitavastatin increased the transendothelial electrical resistance (TEER), an index of barrier tightness of interendothelial tight junctions (TJs), at a concentration of 10^?8 M, and decreased the endothelial permeability for sodium fluorescein through the RBEC monolayer. The increase in TEER was significantly reduced in the presence of isoprenoid geranylgeranyl pyrophosphate, whereas farnesyl pyrophosphate had no effect on TEER. Our immunocytochemical and Western blot analyses revealed that treatment with pitavastatin enhanced the expression of claudin-5, a main functional protein of TJs. Our data indicate that pitavastatin strengthens the barrier integrity in primary cultures of RBEC. The BBB-stabilizing effect of pitavastatin may be mediated partly through inhibition of the mevalonate pathway and subsequent up-regulation of claudin-5 expression.     

FOS／JUN介导佛波酯对内皮素基因表达的诱导作用

利用凝胶电泳迁移率改变实验、RNA印迹和蛋白质印迹分析分别检查了c-jun抗体对内皮素-1(ET-1)基因AP-1位点与核蛋白结合的影响及肿瘤促进剂佛波酯(TPA)对c-fos/c-jun基因表达的作用.结果发现,c-jun抗体可使AP-1位点-核蛋白复合物的电泳迁移率发生改变,TPA显著促进c-fos/c-jun基因表达和血管内皮细胞的AP-1结合活性.实验表明,TPA对ET-1基因表达的诱导作用是通过促进AP-1转录因子c-fos/c-jun合成来介导的.     

Gene Regulation and Functional Alterations Induced by Kaposi's Sarcoma-Associated Herpesvirus-Encoded ORFK13/vFLIP in Endothelial Cells

Kaposi''s sarcoma (KS) is an angioproliferative inflammatory disorder induced by endothelial cell infection with the KS-associated herpesvirus (KSHV). ORFK13/vFLIP, one of the KSHV genes expressed in KS, encodes a 188-amino-acid protein which binds to the Iκb kinase (IKK) complex to activate NF-κB. We examined ORFK13/vFLIP contribution to KS phenotype and potential for therapeutic targeting. Retroviral transduction of ORFK13/vFLIP into primary human endothelial cells induces the spindle morphology distinctive of KS cells and promotes the formation of abnormal vascular networks typical of KS vasculature; upregulates the expression of proinflammatory cytokines, chemokines, and interferon-responsive genes; and stimulates the adhesion of inflammatory cells characteristic of KS lesions. Thymidine phosphorylase, a cellular enzyme markedly induced by ORFK13/vFLIP, can metabolize the prodrug 5-fluoro-5-deoxyuridine (5-dFUrd) to 5-fluouridine (5-FU), a potent thymidine synthase inhibitor, which blocks DNA and RNA synthesis. When tested for cytotoxicity, 5-dFUrd (0.1 to 1 μM) selectively killed ORFK13/vFLIP-expressing endothelial cells while sparing control cells. These results demonstrate that ORFK13/vFLIP directly and indirectly contributes to the inflammatory and vascular phenotype of KS and identify 5-dFUrd as a potential new drug that targets KSHV latency for the treatment of KS and other KSHV-associated malignancies.Kaposi''s sarcoma-associated herpesvirus (KSHV/human herpesvirus 8) is the etiological agent of Kaposi''s sarcoma (KS), primary effusion lymphoma (PEL), and a subset of multicentric Castleman''s diseases. KS typically presents as a multicentric angioproliferative tumor characterized by multiple nodular or macular lesions often on the skin, and less frequently in the gastrointestinal tract and the lung. Histologically, the lesions consist of spindle cells infected with KSHV, inflammatory infiltrates of monocytes/macrophages, lymphocytes and other cells, and “vascular slits” replete of red blood cells (8). KS spindle cells are likely to be of endothelial lineage (19).In KS tissues, KSHV establishes a mostly latent infection characterized by expression of a limited number of viral genes that are likely important to the disease pathogenesis (30). ORFK13 is one such KSHV latent gene. Its gene product, called vFLIP (for viral Flice-like inhibitory protein) or K-FLIP, comprises two tandem death-effector domains that are often found in apoptotic signaling mediators such as cellular FLICE inhibitory protein (cFLIP) and caspase-8/FLICE. Consistent with its sequence similarity with cFLIP, vFLIP was found to inhibit caspase activation and prevent apoptotic cell death (39). Silencing ORFK13/vFLIP expression by RNA interference stopped PEL growth in vitro and in vivo, providing evidence of the essential role of K13/vFLIP in PEL pathogenesis (16). Transgenic mice of K13/vFLIP in lymphoid cells developed more lymphomas than controls (11). Similar to the viral proteins of many other lymphogenic viruses, K13/vFLIP activates NF-κB (1, 10, 25, 27, 42). By activating NF-κB and inhibiting the AP-1 pathway, K13/vFLIP was recently reported to promote viral latency (49).Recent studies have characterized selected effects of K13/vFLIP expression in primary endothelial cells transduced with ORFK13/vFLIP (15, 20, 29), providing important insights into its function. Here, we broadly investigated K13/vFLIP function in endothelial cells. By establishing stable retrovirus-mediated transduction of ORFK13/vFLIP in primary human endothelial cells, we have extensively characterized the biochemical and functional consequences of K13/vFLIP expression in these cells.     

TLR4在四氯化碳诱导的肝硬化大鼠骨髓血窦内皮细胞的表达

目的：检测Toll样受体4（TLR4）在四氯化碳诱导的肝硬化大鼠骨髓血窦内皮细胞的表达,为进一步研究肝硬化时骨髓损伤的发生机制提供实验依据。方法：选择Wistar大鼠给予腹腔注射CCl4,一周两次,建立肝硬化大鼠模型。分别于建模8周和12周检测大鼠血浆内毒素的水平,免疫组化检测大鼠骨髓血窦内皮细胞上TLR4的表达情况,RT-PCR测定骨髓组织中TLR4mRNA的表达,分析TLR4的表达与内毒素血症间的关系。结果：给予CCl4 8周和12周时,对照组大鼠血浆内毒素水平分别为（0．216±0．024）Eu／ml和（0．133±0．022）Eu／ml,模型组大鼠血浆内毒素水平分别为（0．626±0．021）Eu／ml和（0．725±0．031）Eu／ml,分别较对照组显著升高,差异均有统计学意义（P〈0．001）;骨髓血窦内皮细胞TLR4蛋白表达及骨髓组织中TLR4mRNA的表达均显著高于对照组,差异均有统计学意义（P〈0．05）。大鼠骨髓TLR4蛋白和mRNA表达与血浆内毒素水平均呈显著正相关（r=0．841,0．803,P均〈0．001）。结论：CCl4诱导的肝硬化大鼠骨髓血窦内皮细胞TLR4表达升高,并伴随大鼠内毒素血症的发生,提示肝硬化时肠源性内毒素血症可能参与了骨髓的造血功能的损害和病变。     

TLR4 在四氯化碳诱导的肝硬化大鼠骨髓血窦内皮细胞的表达

目的：检测Toll样受体4(TLR4)在四氯化碳诱导的肝硬化大鼠骨髓血窦内皮细胞的表达,为进一步研究肝硬化时骨髓损伤 的发生机制提供实验依据。方法：选择Wistar大鼠给予腹腔注射CCl4,一周两次,建立肝硬化大鼠模型。分别于建模8 周和12 周 检测大鼠血浆内毒素的水平,免疫组化检测大鼠骨髓血窦内皮细胞上TLR4 的表达情况,RT-PCR 测定骨髓组织中TLR4 mRNA 的表达,分析TLR4 的表达与内毒素血症间的关系。结果：给予CCl4 8 周和12 周时,对照组大鼠血浆内毒素水平分别为(0.216± 0.024) Eu/ml 和(0.133± 0.022) Eu/ml,模型组大鼠血浆内毒素水平分别为(0.626± 0.021) Eu/ml 和(0.725± 0.031) Eu/ml,分别较对 照组显著升高,差异均有统计学意义(P<0.001);骨髓血窦内皮细胞TLR4蛋白表达及骨髓组织中TLR4 mRNA的表达均显著高于 对照组,差异均有统计学意义(P<0.05)。大鼠骨髓TLR4 蛋白和mRNA表达与血浆内毒素水平均呈显著正相关（r=0.841,0.803,P 均<0.001）。结论：CCl4 诱导的肝硬化大鼠骨髓血窦内皮细胞TLR4 表达升高,并伴随大鼠内毒素血症的发生,提示肝硬化时肠源 性内毒素血症可能参与了骨髓的造血功能的损害和病变。     

Direct Cytotoxicity of Ethylcholine Mustard Aziridinium in Cerebral Microvascular Endothelial Cells

Abstract: The choline analogue ethylcholine mustard aziridinium (AF64A) is a potent and irreversible inhibitor of choline uptake in brain synaptosomes and is used as a neurotoxin to produce animal models of cholinergic hypofunction. However, previous studies have shown that intraocular administration of AF64A in rats not only reduced the number of cholinergic neurons in the retina, but also induced ultrastructural alterations in the microvasculature. The purpose of this study was to investigate whether AF64A has a direct cytotoxic effect on endothelial cells. As revealed by the measurement of lactate dehydrogenase activity in the culture medium, AF64A produced similar concentration-dependent cellular damage in cultures of bovine cerebral endothelial cells and in the human cholinergic neuroblastoma cell line SK-N-MC, but not in bovine cerebral smooth muscle cells. The toxic effect of AF64A correlated well with the affinity of the choline transport system detected in each cell type. The effect of the toxin on endothelial cells was mediated by its interaction with the endothelial cell choline carrier, as demonstrated by the following observations: (a) AF64A inhibited [³H]choline uptake in a concentration-dependent manner in both cultured and freshly isolated cerebral endothelial cells, and (b) the addition of choline or hemicholinium-3 to the culture medium prevented the AF64A-induced toxicity in endothelial cell cultures.     

过表达KLF4重组质粒的构建及其在白血病K562细胞中的表达

为了构建过表达锌指转录因子Krüppel样因子4(Krüppel-like factor 4,KLF4)基因的重组质粒pEGFP-KLF4,观察其在白血病K562细胞中的表达,以RT-PCR法扩增人KLF4基因,构建pEGFP-KLF4重组质粒;经酶切及测序鉴定后,将pEGFP-KLF4质粒电穿孔转染白血病K562细胞(K562/pEGFP-KLF4组),设pEGFP-C1空质粒转染K562细胞(K562/pEGFP-C1组)及空白K562细胞作为对照,荧光显微镜观察EGFP-KLF4融合蛋白的表达情况,同时用抗生素G418筛选阳性克隆并扩大培养建立稳定过表达KLF4基因的K562细胞株,随后用RT-PCR检测3组K562细胞中KLF4的m RNA表达水平。实验结果显示,pEGFP-KLF4重组质粒构建成功。该质粒转染K562细胞24 h后能观察到较高强度的绿色荧光;经G418筛选出的阳性克隆扩大培养后建立了稳定过表达KLF4基因的K562细胞株;与对照组相比,K562/pEGFP-KLF4细胞组KLF4的m RNA表达水平明显升高,其过表达率为65.71%(P0.05)。实验中构建的过表达KLF4基因的重组质粒pEGFP-KLF4能在K562细胞中高效表达,为进一步研究其在白血病中的作用奠定了基础。     

Direct Evidence of Ion Diffusion for the Silver‐Electrode‐Induced Thermal Degradation of Inverted Perovskite Solar Cells

Perovskite solar cells (PSCs) have recently demonstrated high efficiencies of over 22%, but the thermal stability is still a major challenge for commercialization. In this work, the thermal degradation process of the inverted structured PSCs induced by the silver electrode is thoroughly investigated. Elemental depth profiles indicate that iodide and methylammonium ions diffuse through the electron‐trasnporting layer and accumulate at the Ag inner surface. The driving force of forming AgI then facilitates the ions extraction. Variations on the morphology and current mapping of the MAPbI₃ thin films upon thermal treatment reveal that the loss of ions occurs at the grain boundaries and leads to the reconstruction of grain domains. Consequently, the deteriorated MAPbI₃ thin film, the poor electron extraction, and the generation of AgI barrier result in the degradation of efficiencies. These direct evidences provide in‐depth understanding of the effect of thermal stress on the devices, offering both experimental support and theoretical guidance for the improvement on the thermal stability of the inverted PSCs.     

高糖诱导内皮细胞损伤microRNA表达紊乱的体外研究

目的:研究高糖诱导的内皮细胞损伤微小RNA(microRNA,miRNA)的表达变化。方法:常规培养的人冠状动脉内皮细胞,利用不同浓度D-葡萄糖溶液(0 mmol/L、5 mmol/L、15 mmol/L和25 mmol/L),诱导刺激24 h后分别用CCK-8法和流式细胞术检测其生长活力和凋亡水平。收集细胞总RNA,利用实时定量PCR(Quantitative real-time PCR,q RT-PCR)检测miRNA的表达变化,同时利用TargetScan、PicTar等生物信息学预测软件预测可能的靶基因。结果:高糖溶液(25 mmol/L)刺激内皮细胞后,细胞生长活力明显降低,为对照组的67.5%(P0.01),凋亡水平为对照组的4.5倍(P0.01)。QRT-PCR结果显示miRNA的表达出现了明显的紊乱,其中miR-451、miR-504、miR-302d、miR-18b*、miR-198、miR-328和miR-517c明显下调,miR-29c、miR-100*、miR-137、miR-660和miR-217明显上调(P0.05)。靶基因预测发现miR-217和miR-451可能调控内皮细胞功能相关的多个基因的表达。结论:在高糖诱导的内皮细胞损伤中,miRNA表达紊乱提示其可能参与内皮细胞功能。     

RNA促进小鼠重组染色质白蛋白基因DNaseⅠ消化敏感性

同样的基因在不同的分化细胞中表达不同,基因的选择性表达问题涉及分化和衰老的本质。转录基因对DNaseⅠ(DNA酶Ⅰ)消化敏感,本文研究了RNA对小鼠重组染色质白蛋白基因DNaseⅠ消化敏感性的影响。分离BALB/c小鼠脑细胞核,加入终浓度为2mol/L的NaCl破坏核小体结构,加入不同量、不同来源的RNA,装透析袋,逐渐降低离子强度进行染色质重组。重组染色质中加入DNaseⅠ消化DNA,PCR扩增白蛋白基因的外显子1到外显子2约1200bp区段,PAGE电泳后,用银染色观察不同来源RNA促进DNaseⅠ对白蛋白基因的消化作用。不同组织来源（肝、肺、肾、脑）RNA对小鼠重组染色质中白蛋白基因DNaseⅠ消化敏感性均有促进作用,其中肝和肺RNA促进消化作用较强;酵母tRNA无显著促进消化作用;消化促进作用与RNA剂量有关。RNA能增加DNaseⅠ对白蛋白基因的消化敏感性且有组织（细胞）来源特异性。又委托丹麦Chemical R D 实验室合成2条与白蛋白基因互补的各23核苷酸的RNA,用其进行重组试验。结果表明,重组混合物中含有低至0.2μg/mL的RNA,即可以发挥显著的DNase I消化促进作用。     

直接重整细胞核程序的诱导性多能干细胞研究进展

诱导性多能干细胞（induced pluripotent stem cells, iPS）是分化细胞在外源性因子作用下,经直接细胞核程序重整而重新获得分化潜能的干细胞,具有很重要的应用前景。介绍了iPS诱导方法从转录因子、RNA结合蛋白、小分子化合物、到信号传导通路的发展过程,以及在提高生物安全性方面的改进。iPS的生成在细胞学上表现为渐进的、时间依赖的过程,同细胞的分化状态密切相关;然而,iPS同胚胎干细胞表遗传特征并非完全相同。iPS的进展结合基因治疗和细胞治疗的成果已应用到动物疾病模型的治疗。     

KLF4在内毒素血症小鼠中的表达模式及其意义

目的探讨Kruppel样因子4（Kruppel-like factor 4,KLF4）在内毒素血症小鼠中的表达模式及意义。方法运用实时荧光PCR技术和Western blot技术,分别从mRNA水平和蛋白水平探讨内毒素血症小鼠肝脏和肺脏中KLF4的表达;运用生物信息学技术,对启动子区含有KLF4的结合位点的炎症介质基因进行了预测;运用RT-PCR技术,从mRNA水平探讨内毒素血症小鼠肝脏和肺脏中IL1β的表达模式。结果内毒素血症小鼠肝脏和肺脏中KLF4 mRNA的表达下凋,KLF4蛋白的表达先下凋后升高;IL-18、IL-15、IL-12、IL-18、IL-10等炎症介质基因的启动子区均含有KLF4的结合元件,这些炎症基因的表达可能直接受到KLF4的调控;内毒素血症小鼠肝脏和肺脏中IL-IB的表达模式与KLF4的表达模式呈相反趋势。结论内毒素血症小鼠肝脏和肺脏中KLF4表达下调,KLF4在炎症介质基因表达调控中可能具有重要作用。